CN1062018C - Detecting technique for human haemoglobin (globin) a* gene and gene a* - Google Patents
Detecting technique for human haemoglobin (globin) a* gene and gene a* Download PDFInfo
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- CN1062018C CN1062018C CN92108379A CN92108379A CN1062018C CN 1062018 C CN1062018 C CN 1062018C CN 92108379 A CN92108379 A CN 92108379A CN 92108379 A CN92108379 A CN 92108379A CN 1062018 C CN1062018 C CN 1062018C
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Abstract
The present invention relates to a genetic detection method. A DNA polymerase chain reaction (PCR) technique is utilized, and the deletion of human haemoglobin (globin) alpha 1 genes and alpha 2 genes is detected in the mode of genotyping. Three pairs of amplification primers with definite array order are designed and comprise alpha p 1 and alpha p3, alpha p1 and alpha p 2, and beta p1 and beta p 2 which respectively enlarge Hba1 gene 278 bp, Hba2 gene segment 603 bp and Hb beta gene segment 400 bp. An experimental technique (namely a reagent system of enlargement reaction and a reaction procedure) which is matched with an enlargement reaction is used for enlargement reaction, and the product can be detected by an electrophoresis method. The deletion number of the alpha genes of the human haemoglobin (globin)alpha 1 genes and the alpha 2 genes which are detected can be accurately judged.
Description
The invention belongs to the Genetic Detection field, is to human hemoglobin (globin) α with archaeal dna polymerase chain reaction (PCR) method
1Gene and α
2The technology of gene test.
The α of coding oxyphorase (globin)
1Gene or α
2The disappearance of gene can cause α-Di Zhonghaipinxue disease (it is poor to be called for short ground).It is a kind of common disease in China, and sickness rate is 2.64%, and some areas in Guangxi still do not have effective radical cure method at present unexpectedly up to more than 14.95%, and the patient with severe symptoms needs blood transfusion throughout the year, and not only I am very painful, and family and society are also brought white elephant.
α-ground poor except that minority be cause by α-gene mutations, majority is caused by the disappearance of α-gene.α-gene cluster has 2 α that sequence is very similar
1And α
1Gene, the difference of having only 7 nucleotides sequences to list between the two, they are identical on function, the α-Zhu Danbai molecule of all encoding, the disappearance of these two genes or excalation all can reduce the synthetic of α-Zhu Danbai.In normal people's genome, there are 2 α
1Allelotrope and 2 α
2Allelotrope.It is poor 2 that the patient of a α gene of general disappearance shows as α-ground, and Anemia is very light or do not see symptom; It is poor 1 that the patient of 2 α genes of disappearance shows as α-ground, and Anemia is heavier; The patient of 3 α genes of disappearance shows as the HbH disease, and Anemia is serious, needs blood transfusion throughout the year; The fetus that lacks 4 α genes shows as Bart ' s oedema, can not survive.
Use red blood cell morphology, hemoglobin electrophoresis analysis at present to human hemoglobin (globin) α
1Gene and α
2The disappearance of gene is carried out somatotype and is detected, and also method such as available constraints restriction endonuclease and the hybridization of α gene probe is studied α genetically deficient situation, or these method operative technique complexity, or is difficult to grasp objective standard.Since 1987, generally adopted design at α gene cluster ψ α
1(α
1Pseudogene) intragenic a pair of primer is to human hemoglobin (globin) α
1Gene or α
2The disappearance of gene is carried out PCR (polymerase chain reaction Polymerase chain Reaction) amplification, if the α gene cluster comprises α
1And α
2Gene can obtain correct detected result when interior large fragment deletion; If disappearance only is α
1And α
2Gene or α
1Or α
2Gene and do not comprise ψ α
1The time, then obtain false negative result; If that disappearance only is ψ α
1Gene and α
1And α
2Gene or α
1Or α
2Gene does not lack, and then obtains false positive results.So this PCR detection method has significant limitation, moreover the poor patient in α-ground of China mostly is α
1And α
2Gene or α
1Or α
2Genetically deficient is used this method and is easy to cause detecting mistake.
The objective of the invention is to set up a kind of simple to operate, method is easy, the realizing human hemoglobin of examination criteria (globin) α
1Gene and α
2The pcr gene somatotype detection technique of gene.
The present invention's pcr amplification technology mainly contains that primer contains to become, gene amplification, demonstration detects key step.Invention has designed five kinds of nucleotide primers, and they are:
αp
1：5′d(CGG?CTC?TGC?CCA?GGT?TAA?GG)
αp
1：5′d(CCT?TGG?TCT?GAG?ACA?GGT?AAA?CA)
αp
1：5′d(AGT?GGG?GCC?GAG?GGC?CCA?G)
βp
1：5′d(AAG?GAG?ACC?AAT?AGA?AAC?TGG?GC)
βp
1：5′d(TCT?CCC?CTT?CCT?ATG?ACA?TGA?AC)
α p wherein
1With α p
2Primer amplification Hb α
1Gene segment 278bP, α p
1With α p
2Amplification Hb α: gene fragment 603bp, β p
1With β p
2Amplification Hb beta gene fragment 400bP.Five kinds of primers form three pairs respectively, and the fragment of amplification has obvious characteristics.
Primer α p
1, α p
2, α p
3, β p
1, β p
2By above-mentioned nucleotidesequence, can be synthetic with chemical process.The dna fragmentation of 278bp, 603bp, 400bp can detect with electrophoretic method.As with using ethidium bromide staining after the common agarose gel electrophoresis DNA isolation fragment, the red fluorescence under UV-irradiation detects.
The reagent system of gene amplification is the Tris-HCl (PH=8.1-8.5) that 10-50mM can be arranged in the reaction system of 30-100 μ l, the KCl of 50mM, the NgCl of 0.5-3.0mM
2, (the NH of 10-25mM
4)
2SO
4, gelatin 200 μ g/ml, the first enzyme amine of 4-8%, each 200-300 μ M of dNTP (AGCT), the poly-enzyme, the α p of 0.25-0.5 μ M of containing of the heat-resistant dna of 1-1.5U
1, 0.125-0.25 μ M, α p
1, the α p of 0.125-0.25 μ M
1, the β p of 0.125-0.25 μ M
1, the β p of 0.125-0.25 μ M
1
With mentioned reagent, except hot resistant DNA polymerase, concentrate 10 times, primer is prepared by desired concn, form test kit, then use more convenient.
Adopt above-mentioned PCR reaction reagent system, then the electrophoresis band of pcr amplification product is clear bright, and not assorted band is easy to differentiate.
During amplified reaction, earlier reaction system is mixed beat even, can high speed centrifugation 30 seconds, under 90-93 ℃ of temperature sex change 3-10 minute then, enzyme-added, beat and spare, centrifugal again; Minute do 30-35 circulation by 90-93 ℃-0.5-1 minute, 50-60 ℃ 0.5-1 minute, 68-72 ℃ 1-2; Be incubated several minutes again under 68-72 ℃ of temperature, product electrophoresis on agarose gel plate is answered in negate, observes electrophoresis band under the UV-irradiation.Use Hybaid or FR-300PCR amplification instrument or water bath with thermostatic control amplification, the temperature of employing and soaking time are suitably adjusted in above-mentioned scope.
Amplified production under this amplification program when electrophoresis showed detects, swim be with clear, highly sensitive, with normal people's oxyphorase (globin) α
1Gene and α
2Gene carries out in the product of pcr amplification, and the distance between the electrophoresis band of 278bp, 603bp, three gene fragments of 400bp equates, and is very easy to identify.Mix amplifing reagent system of the present invention and amplification program, these three electrophoresis bands are clear bright, and the brightness of band is close.
With reference to β p
1, β p
2The standard of gene fragment 400bp is if inspected human hemoglobin (globin) α gene is α
1Gene or α
2The heterozygote of genetically deficient, then their α
1Gene or α
2The brightness of the amplified production electrophoresis band of gene fragment only is below half of β gene amplification product 400bp band brightness.
As inspected human hemoglobin (globin) α gene is α
1Gene and α
2The homozygote of genetically deficient then only has the product 400bp band of β gene amplification, and does not have α p
1With α p
3, α p
1With α p
2278bp, the 603bp electrophoresis band of amplification.The α of two sequence similarities of α gene cluster
1Gene and α
2In the gene if the disappearance 1 α
1Gene or α
2Gene then is a heterozygote, and what lack two equipotential α genes is homozygote.
If inspected human hemoglobin (globin) α genetically deficient 3 α genes, then α
1Gene or α
2Gene fragment amplification product electrophoresis band has only a band, and its brightness is below half of β band brightness;
If 4 α genes of inspected human hemoglobin (globin) α genetically deficient do not have α so
1Gene and α
2Gene fragment amplification product electrophoresis band.
Fig. 1 shows the electrophorogram that detects, the 1st, α p
1With α p
3The Hb α of amplification
1Gene fragment 278bp, the 2nd, α p
1With α p
2The Hb α of amplification
2Gene fragment 603bp, the 3rd, β p
1With β p
2The Hb beta gene fragment 400bp of amplification.Blackness is represented the brightness of electrophoresis band among the figure, and row 4 is electrophoresis bands of φ X174 (RF)-Hae III, and row 5 is electrophoresis bands that oxyphorase (globin) α gene does not have disappearance, i.e. the result of normal people's oxyphorase (globin) α gene test, and row 6 is α of disappearance
2Electrophoresis band after the human hemoglobin of gene (globin) the α gene amplification is poor 2 patients' in α ground a detected result, and row 7 is two α gene (α of disappearance
1And α
2) the DNA genome amplification after electrophoresis band, this is poor 1 patient's in α ground a detected result, row 8 be the disappearance 3 α genes the DNA genome amplification after electrophoresis band, this is patient's HbH a detected result, row 9 is the electrophoresis bands behind the DNA genome amplification that all lacks of 4 α genes, and this is Bart ' s oedema patient's a detected result.
Primer order of the present invention is clear and definite, and available dna synthesizer is synthetic automatically, conventional easily row in this research field, and the gene of primer amplification is clear and definite, mixes amplifing reagent system of the present invention, amplification program and electrophoresis detection, makes display result undoubtedly clear.
The present invention not only can detect α respectively
1Gene and α
2Whether gene lacks, and can also compare normal people's inner reference standard, judges that exactly inspected human hemoglobin (globin) α genetically deficient is to belong to α
1Gene and α
2Gene or α
1Gene or α
2The homozygote of genetically deficient or heterozygote.Reliably human hemoglobin (globin) α genetically deficient is carried out somatotype and detect, false positive or false-negative error can not take place detect.
The technology of the present invention is mixed the detection kit made from present technique, and is easy to operate, and detected result is clear and definite, is that the detection of scientific research or α genetically deficient somatotype is all convenient and reliable.
Fig. 1 is human hemoglobin (globin) α
1Gene and α
2The PCR somatotype of gene detects electrophorogram.
Fig. 2 is the pcr gene somatotype detection figure that the HbH disease is not levied earlier person's family.
Embodiment: in 50 μ l systems, contain 10mM Tris-HCl (PH=8.5), 50mMKCl, 2mM MgCl
2, 10mN (NH
4)
2SO
4, gelatin 200 μ g/ml, methane amide 8%, each 200 μ M of dNTP, the α p of 0.25 μ M
1, the α p of 0.125 μ M
2, the α p of 0.125 μ M
1, the β p of 0.125 μ M
1, the β p of 0.125 μ M
2, the about 0.5 μ g of human chromosome DNA is with the topped liquid level of 30 μ l liquid waxes.Beat the reaction system mixing even, high speed centrifugation 30 seconds, 93 ℃ of waters bath with thermostatic control 1 minute add archaeal dna polymerase 1U, beat and spare, centrifugal, 93 ℃ 1 minute, 55 ℃ 1 minute, 68 ℃ of waters bath with thermostatic control are 2 minutes then, carry out 35 circulations successively, 72 ℃ of insulations 5 minutes again after the loop ends, get 20 μ l reaction product electrophoresis on 1.5% sepharose (containing 0.5 μ g/ml EB) plate, observe electrophoresis band under the UV-light.
This person under inspection is the pcr gene somatotype test example that the HbH disease is not levied earlier person's family, observed electrophoresis band result such as Fig. 2.Among this figure the 6th the row (not levying earlier person's father) in the 2nd electrophoresis band be α p
1With α p
2Amplification Hb α
2The brightness of gene segment 603bp band only is below half of brightness of the 3rd β band, can conclude that father is α
2The heterozygote of genetically deficient, what father suffered from is α ground 2 diseases.The 7th row is the result that examined who does not levy earlier mother person, and the brightness that can see the 1st and the 2nd electrophoresis band all is below half of brightness of the 3rd β band, can conclude that mother lacks α
1And α
2Two α gene (α
1And α
2Be heterozygote), thereby suffer from α ground 1 disease.Earlier do not levy person's electrophoretic band and see eighth row, its α
2(603bp) electrophoresis band is completely without, α
1(278bp) brightness of electrophoresis band only is below half of β (400bp) electrophoresis band brightness, can conclude three α genes of its disappearance, suffers from HbH disease.
Claims (1)
1. one kind with archaeal dna polymerase chain reaction technology, to human hemoglobin alpha
1Gene and α
2Genetically deficient is carried out the gene branch
The Genetic Detection method that type detects is characterized in that:
(1) Oligonucleolide primers of gene amplification and order thereof are:
αp
1：5’-CGG?CTC?TGC?CCA?GGT?TAA?GG-3’
αp
2：5’-CCT?TGG?TCT?GAG?ACA?GGT?AAA?CA-3’
αp
3：5’-AGT?GGG?GCC?GAG?GGC?CCA?G-3’
βp
1：5’-AAG?GAG?ACC?AAT?AGA?AAC?TGG?GC
βp
2：5’-TCT?CCC?CTT?CCT?ATG?ACA?TGA?AC；
(2) primer α p
1With α p
3Amplification Hb α
1Gene fragment 278bp, α p
1With α p
2Amplification Hb α
2Gene fragment
603bp, β p
1With β p
2Amplification Hb beta gene fragment 400bp;
(3) the gene amplification reagent system is to contain in 30-100 μ l reaction system:
Tris-HCl：10-50mmol/L?pH=8.1-8.5；KCl：50mmol/L；MgCl
2：0.5-3.0mmol/L；
(NH
4)
2SO
4: 10-25mmol/L; Gelatin: 200 μ g/ml; Methane amide: 4-8%; DNTPs:200-300
Mmol/L; Hot resistant DNA polymerase: 1-1.5U;
αp
1：0.25-0.50μmol/L；αp
2：0.125-0.25μmol/L；αp
3：0.125-0.25μmol/L；
βp
1：0.125-0.25μmol/L；βp
2：0.125-0.25μmol/L；
(4) program of amplified reaction is:
1. 90-93 ℃ sex change 3-10 minute, enzyme-added beat even, centrifugal;
2. press 90-93 ℃ 0.5-1 minute, 50-60 ℃ 0.5-1 minute, 68-72 ℃ 1-2 minute, be 30-35 and follow
Ring:
3. 68-72 ℃ of insulation is after 3-7 minute, and negate answers product containing electrophoresis on the sepharose of EB, ultraviolet
Lamp is observed electrophoresis band down:
(5) detected characteristics is:
1. if inspected human hemoglobin alpha gene is α
1Gene or α
2The heterozygote of genetically deficient, then its α
1Base
Cause or α
2The brightness of the amplified production electrophoresis band of gene fragment only is the bright of oxyphorase β gene electrophoresis band
Spend below half:
2. if inspected human hemoglobin alpha gene is α
1Gene or α
2The homozygote of genetically deficient, then amplified production
During electrophoresis, the β genonema is arranged, do not have α
1Genonema or α
2Genonema;
3. if inspected human hemoglobin alpha gene has lacked 3 α genes, then α
1Gene or α
2Gene fragment expands
Volume increase thing electrophoresis band has only one, and its brightness only is that the brightness of β genonema is below half;
4. if lacked 4 α genes in the inspected human hemoglobin alpha gene, the β genonema is only arranged then, do not have α
1
Genonema and α
2Genonema.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92108379A CN1062018C (en) | 1992-04-14 | 1992-04-14 | Detecting technique for human haemoglobin (globin) a* gene and gene a* |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92108379A CN1062018C (en) | 1992-04-14 | 1992-04-14 | Detecting technique for human haemoglobin (globin) a* gene and gene a* |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1065094A CN1065094A (en) | 1992-10-07 |
| CN1062018C true CN1062018C (en) | 2001-02-14 |
Family
ID=4943489
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92108379A Expired - Fee Related CN1062018C (en) | 1992-04-14 | 1992-04-14 | Detecting technique for human haemoglobin (globin) a* gene and gene a* |
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|---|---|
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063790C (en) * | 1998-09-30 | 2001-03-28 | 复旦大学 | Method for RNA circulation reverse transcription reaction |
| JP2006514547A (en) * | 2002-12-23 | 2006-05-11 | マックス−プランク−ゲゼルシャフト ツール フォーデルング デル ヴィッセンシャフテン エー．ヴェー． | Method for modifying the content of stored substances in plants |
| CN100420943C (en) * | 2005-06-23 | 2008-09-24 | 李卫 | Method for detecting DNA intra-molecularly hybridization with known point mutation - polyacrylamide gel electrophoresis |
| CN111638261B (en) * | 2020-04-17 | 2023-04-07 | 融智生物科技（青岛）有限公司 | Computing equipment, storage medium and thalassemia screening device and system |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1038309A (en) * | 1988-05-27 | 1989-12-27 | 株式会社日立制作所 | Gene tester and device thereof |
| CN1059910A (en) * | 1990-08-15 | 1992-04-01 | 阿斯特拉公司 | A kind of novel method that detects pathogenic agent with dna probe |
-
1992
- 1992-04-14 CN CN92108379A patent/CN1062018C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1038309A (en) * | 1988-05-27 | 1989-12-27 | 株式会社日立制作所 | Gene tester and device thereof |
| CN1059910A (en) * | 1990-08-15 | 1992-04-01 | 阿斯特拉公司 | A kind of novel method that detects pathogenic agent with dna probe |
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| Publication number | Publication date |
|---|---|
| CN1065094A (en) | 1992-10-07 |
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